1. Field of the Invention
This invention relates to and has among its objects the provision of compositions for therapeutic use and methods of making them. It is a particular object of this invention to provide pasteurized compositions containing a concentrate of coagulation Factors II, VII, IX, and X. Further objects of the invention will be evident from the following description wherein parts and percentages are by weight unless otherwise specified.
2. Description of the Prior Art
Many useful blood fractions and blood proteins are obtained from human blood plasma by fractionation according to known techniques such as, for example, the alcohol fractionation method of Cohn described in U.S. Pat. No. 2,390,074 (1945) and the Journal of the American Chemical Society, Vol. 68, page 459 (1946) and the Rivanol.RTM. ammonium sulfate method. The aforementioned methods as well as other variations and techniques are summarized in "The Plasma Proteins", second edition, Volume III, pages 548-550, Academic Press, New York, NY (1977). These blood fractions contain biologically active proteins that possess certain therapeutic qualities. For instance, a concentrate of Factors II, VII, IX and X is useful in treating individuals who have hemophilia. There are estimated to be 100,000 cases of congenital hemophilia in the U.S. Of these, approximately 20,000 are cases of hemophilia B, the blood of such patients being either totally devoid of plasma thromboplastin component or seriously deficient in plasma thromboplastin component. The disease therefore exists in varying degrees of severity, requiring therapy anywhere from every week up to once or twice a year. The completely deficient cases require replacement therapy once every week; the partially deficient cases require therapy only when bleeding episodes occur, which may be as seldom as once a year. The bleeding episodes in congenital, partially-deficient cases are generally caused by a temporarily acquired susceptibility rather than by injury alone. Intravenous injection of a sufficiently large amount of fresh plasma, or an equivalent amount of fresh blood, temporarily corrects the defect of a deficient subject. The beneficial effect often lasts for two or three weeks, although the coagulation defect as measured by in-vitro tests on the patient's blood appears improved for only two or three days. Such therapy with fresh plasma or fresh blood is effective but it has several serious drawbacks: (1) it requires ready availability of a large amount of fresh plasma; (2) requires hospitalization for the administration of the plasma; (3) a great many of the patients become sensitized to repeated blood or plasma infusions and ultimately encounter fatal transfusion reactions; (4) at best plasma can only partially alleviate the deficiency; and (5) prolonged treatment or surgery is not possible because the large amounts of blood or plasma which are required will cause acute and fatal edema.
Because of the above, concentrates of coagulation Factors II, VII, IX, and X (Factors IX concentrates) have been developed for administration to hemophilia B patients (for example, the concentrate of Factors II, VII, IX, and X disclosed in U.S. Pat. No. 3,717,708).
One problem confronting users of Factor IX concentrates is the thermal instability of the therapeutically active proteins contained therein. In many cases, substantial, and sometimes complete, losses of activity are observed if these concentrates are heated above physiologically compatible temperatures, i.e., above about 40.degree.-45.degree. C. Consequently, these items require special care during preparation and storage to minimize such deactivation.
The thermal instability of the aforementioned proteins renders them unpasteurizable. Therapeutically active proteins isolated from plasma may contain viruses, e.g., hepatitis virus, present in the source material for the protein fraction, namely, blood from a donor. A risk of contracting hepatitis exists, therefore, for those receiving unpasteurized fractions from blood plasma fractionation because the presence of the virus cannot be detected with certainty by any known procedure. In a large number of situations, this risk is outweighed by the detriment to a patient in not receiving the therapeutic plasma fraction as determined by the physician.
Some therapeutically active proteins derived from plasma have been pasteurized successfully. For example, it is well known that albumin can be pasteurized by heating at 60.degree. C. or 64.degree. C. for 10 hours (Gellis et al, J. Clin. Invest., Vol. 27, pages 239-244 [1948]) in the presence of certain stabilizers such as acetyl-tryptophan and sodium caprylate. Individuals receiving this pasteurized material did not contract hepatitis, thus indicating the inactivation of hepatitis virus while retaining the activity of albumin under the afore-described heating conditions. Plasma Protein Fraction (human) is also stabilized during pasteurization by the above method.
A process for pasteurizing plasminogen is disclosed by Baumgarten et al in U.S. Pat. No. 3,227,626. An aqueous preparation containing 0.25-20 milligrams per milliliter (mg/ml) of plasminogen and further containing 0.1-0.5 molar lysine with a pH of 5.3-7.5 was heated at 60.degree. C. for 10 hours. As the patentee states, hepatitis virus was destroyed and the danger of transmitting hepatitis was removed with retention of plasminogen activity. Attempts to pasteurize plasminogen under the above conditions in the absence of lysine resulted in complete destruction of plasminogen activity. It is interesting to note that plasminogen cannot be stabilized with N-acetyl-tryptophan and sodium caprylate during pasteurization, nor can albumin and Plasma Protein Fraction (human) be pasteurized in the presence of lysine.
Singher has described a process for treating plasminogen to produce a material that is not contaminated with hepatitis virus (U.S. Pat. No. 2,897,123). In the patented pasteurization technique aqueous solutions of plasminogen are heated at about 60.degree. C. for about 10 hours. The activity of plasminogen is retained if the solutions have a pH in the range not less than 3 nor greater than 6.5 and an ionic strength not greater than 0.2.
Another method for removing hepatitis virus from a biological material is described in U.S. Pat. No. 4,168,300. The material to be treated is contacted with a preparation, which may be agarose gel or beaded polyacrylamide plastic coupled with a variety of hydrophobic ligands. Plasma and albumin were subjected to the above purification technique to remove hepatitis virus.
Aqueous solutions of the enzyme thrombin have been stabilized (Seegers, Arch. Biochem., 1944, Vol. 3, pages 363-367) during heating at 50.degree. C. in the presence of saturation amounts of certain glycosides. The stabilized solutions were heated at the above temperature for a period of 48 hours or more with minimal loss of activity. On the other hand, Seegers also discloses that glycosides and polyols have only minimal effectiveness in stabilizing the enzyme prothrombin. The reversible denaturation of lysozyme and ribonuclease was studied by Gerlsma et al, Int. J. Peptide Protein Res., Vol. 4, pages 377-383 (1972). The authors found that certain polyhydric alcohols increased somewhat the temperatures at which these enzymes were denatured. Finally, Simpson et al, in J. Am. Chem. Soc., Vol. 75, No. 21, pages 5139-5152 (1953) and Donovan in J. Sci. Fd. Agric., Vol. 28, pages 571-578 (1977) noted that the denaturation temperature of ovalbumin (an egg white protein) was raised slightly in the presence of sucrose in aqueous solutions of the protein. However, Donovan points out that the temperatures of denaturation of ovalbumin and S-ovalbumin are 84.5.degree. C. and 92.5.degree. C., respectively. Furthermore, ovalbumin and S-ovalbumin, as well as the aforementioned enzymes, have no therapeutic activity in treating disorders in humans, whereas blood plasma proteins are therapeutically active. In fact, as mentioned below, proteolytic enzymes deactivate blood plasma proteins.
Singher, in the aforementioned U.S. patent, lists some methods of destroying hepatitis virus. The least effective of these methods involves the use of either nitrogen mustard or beta-propiolactone. High energy irradiation in appropriate dosage is effective but destroys biological activity when applied to human blood products. Heat is recognized also as effective against hepatitis virus, the preferred treatment being heating the material at 60.degree. C. for 10 hours. Higher temperatures above 70.degree. C. for shorter intervals or lower temperatures for longer intervals have also been tried with successful results. However, it is important to note that higher temperatures are undesirable because of the potential for denaturation of the proteins. Furthermore, lower temperatures for long intervals are to be avoided because various proteolytic enzymes are activated under these conditions, and these activated enzymes cause protein degradation. Also, the use of temperatures lower than 60.degree. C. for pasteurization has not been shown to consistently yield a material that does not contain the infective virus.
As mentioned above, the recognition that heating at 60.degree. C. and 64.degree. C. for 10 hours successfully destroys the hepatitis virus in albumin was made by Gellis et al, supra. Gellis et al proved experimentally that albumin heated under the above conditions did not transmit hepatitis even if hepatitis virus was present prior to pasteurization. However, the author noted that hepatitis virus survived heating at 56.degree. C. for one hour, a temperature usually employed for the inactivation of viruses. Thus, although heating at temperatures of about 56.degree. C. for one hour will deactivate most viruses, hepatitis virus is not inactivated; and materials containing hepatitis virus, which are heated at 56.degree. C. for one hour, cause infection of hepatitis in individuals receiving such materials.
Japanese Pat. No. 51-134878 (1976) teaches the stabilization of Factor XIII against heat inactivation (60.degree. C. for 10 hours) by using 10-20% (w/v) of a stabilizer such as a neutral amino acid, a monosaccharide, or a sugar alcohol. Furthermore, in U.S. Pat. No. 4,297,344 there is disclosed a method of stabilizing coagulation Factors II, VIII, XIII, antithrombin III, and plasminogen against heat in the presence of a 1-3 molar amount of a certain amino acid and 20-60% (w/w) of a carbohydrate. Haptoglobin has been pasteurized in the presence of a stabilizer such as an amino acid, a mono- or di-saccharide or a sugar alcohol.
In German Offenlegungsschrift No. 3043857 there is disclosed a method for the manufacture of a hepatitis-free preparation of coagulation Factors II and/or VII. The method is characterized by mixing the preparation of Factor II and/or VII with an amino acid and/or a saccharide or sugar alcohol and a chelating agent such as ethylene diamine tetraacetate or theylene glycol-bis-(2-aminoethylene either)-tetracetate and heating the mixture to inactivate hepatitis virus.
Antithrombin compositions have been mixed with citrate ions in order to stabilize the antithrombin to heat. (Holleman et al, Thromb. Haemostasis, 38, 201 [1977]).